C E L L BIOCHEMISTRY AND FUNCTION VOL.

10: 185-191 (1992)

Transcriptional Regulation of Genes Encoding Proteins Involved in Biogenesis of Peroxisomes in Saccharomyces cerevisiae A. W. C. EINERHAND, I. VAN DER LEIJ, W. T. KOS, B. DISTEL AND H. F. TABAK1E.C. Sluter. Institute fbr Biochemical Research, University of Amsterdam, Meihergdreej 15, 110.5 AZ Amsterdum, The Nc~rherlundk

INTRODUCTION Peroxisomes constitute a dynamic subcompartment of the eukaryotic cell that can be recruited In when their enzymatic repertoire is required. higher eukaryotes the biogenesis of peroxisomes is dependent on the differentiation that cells undergo: liver cells contain numerous peroxisomes while in erythrocytes they are absent. Furthermore, in rodents certain xenobiotics can stimulate proliferation of peroxisomes, underscoring the adaptive capacity of this ~ r g a n e l l e .This ~ proliferation has been tuned to near-perfection by certain fungi such as the methylotrophic yeasts. When grown on a carbon or nitrogen source (which require peroxisoma1 functions for their metabolism), these yeasts respond by dramatically increasing their peroxisoma1 c ~ r n p a r t m e n t .An ~ important acceleration in this field of research took place when conditions were discovered that stimulate peroxisome proliferation in Succliuromyces ~erevisiae.~ The long history of basic research in this yeast has resulted in a wealth of information about growth control under various conditions. Studies on the function and biogenesis of peroxisomes in S. cerevisiae therefore could benefit from this knowledge. The proliferation of peroxisomes is most likely controlled at the transcriptional level, since it has been shown recently that several Saccharomyces wretisicie genes encoding peroxisomal proteins are regulated mainly at the level of The contours of the cis-acting DNA elements, trans-acting transcription factors and components of signal transduction networks that are involved are becoming clear. It is a challenge to investigate

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how peroxisome proliferation fits in with the signal transduction pathways mediating glucose and nitrogen catabolite repression.’-’ l Two approaches are beginning to yield insight into the control of peroxisome proliferation in yeast. Firstly, promoters belonging to genes coding for peroxisomal proteins such as catalase A and 3-oxoacyl-CoA thiolase are being studied with respect to their transcriptional control. Secondly, mutants have been isolated that have defects in their capacity to grow on oleate, a carbon source that can only be used for growth when functional peroxisomes are present. Here we will discuss, as a representative example, mainly the work concerning the thiolase promoter, including our recent results. In addition, evidence that stems from studies of the catalase A promoter is also presented. In combination, these results enable us to present a rough outline of signal transduction pathways influencing the biogenesis of peroxisomes.

TRANSCRIPTIONAL CONTROL ELEMENTS AND TRANS-ACTING FACTORS In order to measure the output of the thiolase gene, the luciferase coding region was fused in frame shortly after the AUG initiation triplet. Luciferase can be measured at very low levels and is stable in crude yeast extracts. Three different states of transcription can be observed when cells are cultivated under various growth conditions (Figure 1): Expression is almost completely suppressed in the presence of high glucose (glucose repression); limited expression results from growth on glycerol

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.-C I GLUCOSE

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GLYCEROL OLEATE

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thiolase promoter

Thiolase Dromoter activitv: glucose growth conditions (repression): glycerol growth conditions (derepression): oleate growth conditions (induction):

promoter is switched off promoter is weakly active promoter is fully active

Figure 1. The peroxisomal thiolase expression levels. Luciferase activities mediated by the thiolase promoter-luciferase fusion (yTL98S6) in response to glucose (repression), glycerol (derepression) or oleate (induction). Activity measured in crude extract obtained from cells grown on oleate, was taken as 100 per cent.

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TAATGATGTG GTAGCGCCGT GTAAGGCGCT ATCAAAGGGA AACGGGGATA ATAGTAmAA CACCGCAGCT TTTTTTI‘CCT TTCTCCCTCT AlTGGTTTCA AATTTATTGG AGTTT ATTACTACAC CATCGCGGCA CATTCCGCGA TAGTTTCCCT TTGCCCCTAT TATCATAATT GTGGCGTCGA AAAAAAiiGGA AAGAGGGAGA TAACCAAn’GT T T A U T A A C C T C W

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Figure 2. Transcriptional control elements of the thiolase promoter. Nucleotide sequence between -230 and - 116 (relative to the translational initiation codon) of the thiolase gene is presented. Below a schematic representation is given of the different cis-acting elements based on sequence homology in addition to experimental results (for detailed description of these sites see text). The FUFl (FOX3 Upstream Factor) site indicates the sequence motif similar to the CAR1 URS. Arrows indicate an imperfect inverted repeat.

(derepression); full induction occurs during growth on oleate (induction) as the sole carbon source.6 In order to achieve this tuning of gene expression, the thiolase promoter contains a number of cis-acting elements to which various proteins can bind. Figure 2 presents a summary of our present knowledge which is based on: Promoter deletion studies; gel retardation analysis; DNase I footprint analysis; in

vivo determination of protein-DNA contacts (together with w. Mulder and L. A. Grivell, Section for Molecular Cell Biology, University of Amsterdam); site-directed mutagenesis experiments and DNA sequence Sequences upstream of -238 (relative to the translational initiation codon of the thiolase gene) can be deleted without significantly affecting ex-

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TRANSCRIPTIONAL REGULATION OF GENES ENCODING PEROXISOMAL PROTEINS

pression of the reporter gene. However, deleting protein(s) binding to the URS of the CAR1 gene sequences upstream of -203 results in an increase has still to be ascertained. Further deletion (up to-188) causes a loss of of luciferase activity in cells grown on glucose, glycerol o r oleate. This increase is most pro- induction capacity and results in only a basic level nounced in cells grown on glucose, suggesting of transcription. On the basis of this result and of release from glucose repression. A DNA fragment DNA sequence comparison (see also Figure 3 ) an (from 238 to - 196) incubated with an unfrac- oleate response element (ORE) was defined (pretionated yeast protein extract gives rise to two viously referred to as p-oxidation box)6 which is protein-DNA complexes in a band-shift assay. required for oleate induction. This was substanDNA sequence comparison indicates the presence tiated by two different in uivo experiments. Firstly, a of an ABFl site, and E. coli-produced ABFl does DNA fragment containing only the ORE element, indeed bind to this DNA sequence motif fused to a UAS-less heterologous promoter, mespecifically.6 ABFl (ARS Binding Factor) is an diated induction of transcription in response to abundant yeast factor binding to the 5'-flanking oleate. Secondly, mutations in the ORE element in regions of many different genes and to ARS (Au- the homologous thiolase promoter context negatitonomous Replicating Sequences) and therefore is vely affected the response to oleic acid. Gel retardathought to play a general role in transcriptional tion analysis demonstrates that the ORE element regulation and in DNA r e p l i ~ a t i o n . ' ~ -The ' ~ func- serves as a protein binding site. However, in citro tion of the ABFl binding site in the thiolase and in vico footprint analysis indicate that propromoter context is still unknown, since a mutation tein(s) bind not only to the ORE element but also in the ABFl binding site that reduces drastically in I I I I I I I citro DNA binding of ABF1, has little effect on the I promoter output in uico. However, the ABFl site is located in a nuclease-hypersensitive DNA region CTAl indicating that this region is free of nucle~somes.'~ Therefore, it is tempting to speculate that ABF1, in analogy with the function of another multifuncT FOX3 tional yeast protein, RAP1 18, might be involved in the positioning of nucleosomes in this context. Partially overlapping the ABFl binding site, a sequence motif is located which is strikingly similar to the consensus DNA sequence established by Cooper and collaborators.' 9 . 2 0 They showed that this DNA element, located in front of the CAR1 gene which encodes arginase, is a protein-binding T PASI site required to repress transcription in the absence of the inducer, arginine. Furthermore, they showed @Fj that the upstream region of the yeast peroxisomal catalase A gene, and, in addition, 13 other unrelated yeast genes, contain sequences similar to the CAR1 URS (upstream repression site). These 14 sequences, resembling the CAR1 URS, were deT Lmonstrated to bind protein and to compete with a DNA fragment containing the CAR1 URS for .. LOX3vpstream factor hindin8 'itc protein binding. Moreover, these sequences were @ A B F l binding site /FUFi \ ~ ~ , shown to support varying degrees of URS function Qleate Kesponse Element T TATA box in an expression vector A DNA fragment (from - 238 to - 196) obtained from the 5'A D R l binding site flanking region of thiolase that no longer binds ABFI, still forms a DNA-protein complex in a Figure 3. Modularity of cis-acting elements. Putative cisband-shift assay. The protein(s) giving rise to this acting elements present in the 5' Ranks of the S. cerevisiue genes encoding pe:.oxisomal matrix (CTAI, FOX1,2,3 and CIT2) or complex might be responsible for negative trans- membrane ( P A S 3 ) proteins or encoding a protein involved in criptional control. However, its homology with the peroxisomal assembly ( P A S I ) . ~

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immediately downstream of the ORE motif. The DNA sequence shows the presence of an inverted repeat and it may well be that this forms the main target site for the transcription factor(s) mediating induction. Finally, a DNA sequence resembling an A D R l binding site is located between - 151 and - 116. ADRl (Alcohol Dehydrogenase I1 synthesis Regulator) is a transcriptional activator, which was first identified genetically as a factor required for the synthesis of alcohol dehydrogenase I1 from S. cerevisiae2'922A CAMP-dependent protein kinase phosphorylates ADR 1 and thereby inactivates it under glucose-repressed condition^.^^ By Northern blot analysis Ruis and co-workers have shown that ADRl is also required for derepression of the genes

encoding catalase A (CTAI), the multifunctional poxidation enzyme (FOXZ),thiolase (FOX3) and the PAS1 (peroxisome assembly) gene product.8 Gel retardation analysis demonstrated that ADRl binds to a catalase A upstream fragment ( - 184 to - 156). Furthermore, the observation that the expression of the catalase A gene is reduced in a bcyl mutant with high unregulated protein kinase A activity provides an interesting parallel to the influence of this mutation on the expression of alcohol dehydrogenase 11.8 The importance of the cis-acting DNA elements identified in the thiolase promoter is underscored by the observation that corresponding sequence elements are often found in other genes coding for peroxisomal protein^^,',^^,^^ or proteins essential

high glucose

Figure 4. Signal transduction pathways influencing peroxisome biogenesis in yeast. Repression of transcription by glucose is mediated either via the CAMP-dependent protein kinase pathway reaching ADRl in the nucleus (grey shaded area) or via a pathway involving FUFl ( F O X 3 Upstream Factor) which binds to the sequence sharing homology to the CAR1 URS. Derepression is mediated via the cytosolic SNF1. This signal could be further transmitted to SNFZ, SNF5 and SNF6 in the nucleus. The oleate induction signal pathway via unidentified intermediates in the cytoplasm reaches the ORE binding factor (ORE bf) in the nucleus.

TRANSCRIPTIONAL REGULATION OF GENES ENCODING PEROXISOMAL PROTEINS

for peroxisome a ~ s e m b l y ~(Figure ~ , ~ 3). Exceptions thus far are the PAS3 gene, which codes for an integral peroxisomal membrane protein,” and the CIT2 gene which codes for citrate ~ y n t h a s eThe .~~ PAS3 gene contains only an ORE element and in the CIT2 gene neither of these DNA elements can be discerned. However, it has been shown that synthesis of peroxisomal membrane proteins precedes synthesis of matrix proteins after induction of peroxisome proliferation in organisms as widely divergent as the rat28 and the yeast Candida boidinii.” Therefore, it is conceivable that transcription of the PAS3 gene is controlled in a somewhat different manner and has a slightly different configuration of &-acting elements compared to most of the genes coding for matrix proteins listed in Figure 3. Transcriptional control of the CZT2 gene may be different altogether, since it is dependent on unspecified mitochondria1

function^.^' MUTANTS DEFECTIVE IN PROLIFERATION OF PEROXISOMES The requirement of the ADRl protein for derepression identifies a signal transduction route involving a CAMP-dependent protein kinase in peroxisome proliferation (Figure 4). In this case there is evidence that ADRl is always bound to its DNA binding site but that the phosphorylation state determines its function, de-phosphorylation being required for d e r e p r e ~ s i o n . ~Recent ’ . ~ ~ results using immuno-cytochemistry indicate that wild-type yeast cells grown on glycerol contain more peroxisomes (visualized using anti-thiolase and gold par-

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ticles) than adrl-mutant cells grown under the same conditions. However, the number of peroxisomes increases in both wild type and adrl-mutant cells grown on oleate suggesting, in agreement with results reported by Ruis and colleagues,’ that the oleate induction is unaffected in this mutant. This indicates that derepression (via ADR 1) and oleate induction represent different and independent signal transduction pathways (see Figure 4). Selection of S. cereoisiae mutants disturbed in peroxisome assembly and/or proliferation has resulted in a number ofpas mutants with abnormalities in the induction of transcription of genes coding for peroxisomal proteins (see also the review by Erdmann and Kunau, this issue). The pasl4- mutation negatively affects the in vitro binding of protein to the ORE element of the thiolase gene.33 The gene complementing the pasl4- mutant has been cloned and sequenced and has turned out to be the same as that of SNFI, which codes for a cytosolic serine/threonine protein kinase already described and studied by Carlson and c o - ~ o r k e r s It . ~is~ required for the activation of many glucose-repressible genes and can be taken as a positive regulator for derepression of transcription when cells are grown on non-fermentable carbon sources.35 The signal transduction route reaches the nucleus via unknown intermediates. Here it could be that proteins specified by the genes SNF2, SNF5 and SNF6 transmit the signal further (Figure 4). The observation that s n j T , snfs- and snf6- mutants, like snfl-, do not grow on oleate is in line with this interpretation. The phenotypes of s n j T = swi2-, snf5- and snf6- mutants are, in general, strikingly similar to those exhibited by

Figure 5. As a representative example the configuration of cis-acting elements present in the thiolase gene is drawn schematically. The complex of SWI1, SWIZ/SNF2, SWI3, SNF5 and SNF6 could be involved in the induction of transcription by oleate by acting as a bridge between the ORE binding factor (ORE bf) and the pre-initiation complex. For details see text.

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swil- and swi3- mutants, indicating that SWI1, eds.) Academic Press: London, pp. 601 -653. SWI2/SNF3, SWI3, SNF5 and SNF6 are all re- 5. Veenhuis, M., Mateblowski, M., Kunau, W.-H. and Harder, W. (1987). Proliferation of microbodies in Sacquired for transcription of the same large set of charomyces cerevisiae. Yeast, 3, 77-84. genes.36337The nuclear proteins SNF2, SNFS and 6. Einerhand,A. W. C., Voorn-Brouwer, M. M., Erdmann, R., SNF6 do not bind to DNA themselves but are Kunau, W.-H. and Tabak, H. F. (1991). Regulation of transcription of the gene coding for peroxisomal 3-oxoasupposed to function in a very large multi-subunit cyl-CoA thiolase of Saccharornyces cerevisiae. Eur. J. Biocomplex36 together with the proteins specified by chem., 200, 113-122. the SWIl and SWI3 genes which also do not bind 7. Dmochowska, A., Dignard, D., Maleszka, R. and Thomas, to DNA directly.37 Herskowitz and c o - ~ o r k e r s ~ ~ D. Y. (1990). Structure and transcriptional control of the speculate that such a large complex might be Saccharomyces cerevisiae POX1 gene encoding acyl-Coenzyme-A-oxidase. Gene, 88, 247-252. capable of providing enough diversity to interact 8. Simon, M., Adam, G., Rapatz, W., Spevak, W. and Ruis, H. with and assist many different gene-specific activa(1991). The Saccharomyces cerevisiae A D R l gene is a tors. Based on this assumption, a speculative model positive regulator of transcription of genes encoding peroxof the function of the SWII, SWI2/SNF2, SWI3, isomal proteins. Mol. Cell. Biol., 11, 699- 704. 9. Cooper, T. G. (1982). Nitrogen metabolism in SaccharoSNFS and SNF6 complex in relation to oleateni.yces cerecisiae. In: Molecular Bioloyy of the Yeast Sacinduced transcription of genes encoding proteins churomyces: Metaholism and Gene E.xpression. (Strathern, involved in peroxisome biogenesis is illustrated in H., Jones, E. and Broach, J., eds.) Cold Spring Harbor Figure 5. Laboratory: Cold Spring Harbor, N.Y., pp. 39-99. 10. Trumbly. R. J. (1992). Glucose repression in the yeast

CONCLUDING REMARK A characteristic feature of the thiolase promoter to emerge from the studies described here is the modularity of promoter elements. Mixing a limited number of regulatory sites and associated factors in promoter regions would provide a conceptual basis for the integration of multiple environmental regulatory signals, such as the presence of oleate, and low or high levels of glucose in the growth media.

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ACKNOWLEDGEMENTS We are grateful to Professors P. Borst and W.-H. Kunau, Drs R Benne and P. Sloof for stimulating discussions and to colleagues in the field for providing us with some of the strains and mutants used for our studies. We thank A. Motley for her help to improve the English text. This work was supported by the Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Foundation for Scientific Research (NWO) and the Biotechnology Center Amsterdam.

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